老年大鼠脊髓小胶质细胞分选新方法及功能特征观察*

doi:10.12047/j.cjap.6194.2022.017

中国应用生理学杂志 ›› 2022, Vol. 38 ›› Issue (1): 91-96.doi: 10.12047/j.cjap.6194.2022.017

• 技术方法 • 上一篇

老年大鼠脊髓小胶质细胞分选新方法及功能特征观察^*

张雨彤, 赵欣^△, 尹艺儒, 吴淑芬, 张利娜, 杜相欣, 张宇^△

山西医科大学生理学系细胞生理学教育部重点实验室, 太原 030001

收稿日期:2021-04-03 修回日期:2022-01-04 出版日期:2022-01-28 发布日期:2022-05-30
通讯作者: ^△Tel: (0351)4135560, 3985163; E-mail: zhyucnm@163.com, xinzhao2006@163.com
基金资助:
* 国家自然科学基金项目(81371254);山西省‘1331工程’重点学科建设计划经费(XK201708);山西省自然科学基金(201801D121318);山西省研究生创新项目(2020SY222);山西省省筹资金资助回国留学人员科研项目(2021-080)

A new method of microglia sorting and functional characteristics of spinal microglia in aged rats

ZHANG Yu-tong, ZHAO Xin^△, YIN Yi-ru, WU Shu-fen, ZHANG Li-na, DU Xiang-xin, ZHANG Yu^△

Key Laboratory of Cellular Physiology, Ministry of Education; Department of Physiology, Shanxi Medical University, Taiyuan 030001, China

Received:2021-04-03 Revised:2022-01-04 Online:2022-01-28 Published:2022-05-30

摘要/Abstract

摘要： 目的: 建立分离纯化老年大鼠小胶质细胞的改良方法,并初步观察老年大鼠脊髓小胶质细胞的生物学特性。方法: 以年轻SD大鼠(2月龄)为对照组,采用胰酶、胰酶替代物和机械网搓法等不同的制备方法,制备大鼠小胶质细胞的单细胞悬液,通过检测细胞纯度、存活率,观察细胞形态特征,分析细胞的炎性功能特征等,确定老年大鼠(20月龄)小胶质细胞的分离纯化方法,观察老年大鼠脊髓小胶质细胞功能特征。结果: 胰酶消化所得细胞的存活率低(年轻大鼠83%,老年大鼠60%);机械网搓法虽得到的存活率较高(95%),但是细胞获取率最低(年轻大鼠((0.207±0.020)×10⁶,老年大鼠(0.243±0.023)×10⁶);采用胰酶替代物解离、密度梯度离心方法分选出的老年大鼠脊髓小胶质细胞数量多、活性好、存活率高,细胞纯度可达85%以上,我们采用此方法分选纯化不同年龄大鼠脊髓小胶质细胞,与年轻大鼠相比,老年鼠脊髓组织量大,所需消化液多,但消化时间缩短;与年轻大鼠小胶质细胞相比,老年大鼠脊髓小胶质细胞其胞体较大较圆,突起少且粗短,形态上偏向于激活状态,老年大鼠小胶质细胞促炎因子IL-1β表达降低(P＜0.05),而抗炎因子IL-10(P＜0.01)表达升高。结论: 成功建立胰酶替代物解离结合密度梯度离心法从大鼠脊髓组织中分离纯化小胶质细胞,老年大鼠脊髓内小胶质细胞整体表现出抗炎表型。

关键词: 老年大鼠, 脊髓, 小胶质细胞, 分离纯化, 炎性因子

Abstract: Objective: To establish an improved method of separating microglia from aged rats and to observe the biological characteristics of spinal microglia of aged rats. Methods: Young SD rats (2 months) were used as control group. Single cell suspension of rat microglia were prepared by trypsin, trypsin substitutes or mechanical net rubbing method. Then, by assessing the purity and survival rate of cells, and observing the morphological characteristics and analyzing the inflammatory functional characteristics, we optimized the isolation and purification method of microglia from aged rats (20 months old) , and observed the functional characteristics of spinal microglia in aged rats. Results: The survival rate of cells digested by pancreatic enzyme was low(young rats 83%, aged rats 60%). Although the survival rate of mechanical net rubbing method was higher than that of pancreatic enzyme digest methods (95%), the cell acquisition rate was lower(young rats(0.207±0.020)×10⁶, aged rats(0.243±0.023)×10⁶). Trypsin substitute dissociation combining density gradient centrifugation method was the best way to get abundant, active and higher survival microglia, and the purity reached more than 85%. We used this method to separate microglia from spinal cord of rats. Compared with the young rats, the spinal cord tissue of old rats was larger, the digestive fluid volume was higher, but the digestion time was shorter. Compared with the young rats, the aged rat spinal microglia had larger and rounder cell body, fewer and shorter protrusions, it tended to be activated morphologically, the level of proinflammatory cytokine IL-1β of microglia in aged rats was lower, and the level of antiinflammatory factor IL-10 was higher. Conclusion: The method of trypsin substitute dissociation combined with density gradient centrifugation was successfully established to isolate and purify microglia from spinal cord of rats, the spinal microglia of old rats showed anti-inflammatory phenotype.

Key words: aged rat, spinal cord, microglia, isolation and purification, inflammatory factors

中图分类号:

R338.3

张雨彤, 赵欣, 尹艺儒, 吴淑芬, 张利娜, 杜相欣, 张宇. 老年大鼠脊髓小胶质细胞分选新方法及功能特征观察^*[J]. 中国应用生理学杂志, 2022, 38(1): 91-96.

ZHANG Yu-tong, ZHAO Xin, YIN Yi-ru, WU Shu-fen, ZHANG Li-na, DU Xiang-xin, ZHANG Yu. A new method of microglia sorting and functional characteristics of spinal microglia in aged rats[J]. CJAP, 2022, 38(1): 91-96.

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老年大鼠脊髓小胶质细胞分选新方法及功能特征观察^*

A new method of microglia sorting and functional characteristics of spinal microglia in aged rats

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